Chemical and biological approaches to enhance the bacteriophage as a probe in molecular recognition

The aim of the work presented in this thesis was to explore the structural and molecular features of a filamentous bacteriophage for incorporation in biosensing platforms. To tune the bacteriophage surface as a scaffold for molecular recognition, chemical and biological protocols aimed at controlling and enhancing its intrinsic properties were developed. A key part of the work was aimed at anchoring bacteriophage particles by one-end to surfaces, and on labelling its viral capsid with dyes, antibodies and DNA molecules. The tolerance of the bacteriophage for modifications, as well its simplicity and robustness, makes it an attractive probe for biosensors. Furthermore, the filamentous structure of the bacteriophage was demonstrated to be a valuable feature in both microscopy- and spectroscopy-based biosensors. The ability of the bacteriophage to align under flow was considered as a simple, yet efficient approach for the detection of wall shear stress and pathogenic bacteria. A novel method to detect wall shear stress (WSS) was developed by using a fluorescently decorated bacteriophage particle anchored by one-end to a surface. The response of this filamentous nanosensor to flow variations was tracked under a microscope, and gave valuable information on the shear flow of a fluid passing over a surface. Using a custom-made analysis tool and an algorithm, we were able to derive the wall shear stress on the point of attachment of the nanosensor, using endothelial cells as a model system. The proof-of-concept to this work highlighted how a simple bacteriophage construct can be use as a nanosensor for imaging and mapping flow. Linear dichroism (LD) spectroscopy also explores the high aspect ratio of the filamentous bacteriophage. Linear Diagnostics Ltd., a startup company in Birmingham, exploits the natural LD properties of the bacteriophage and integrated it with a biosensing platform targeting pathogenic bacteria. The work reported in this thesis mainly focused on developing alternative approaches that could improve the biosensor sensitivity and simplicity, by modifying the bacteriophage scaffold, further demonstrating its versatility

Prospective, randomized trial comparing fluids and dobutamine optimization of oxygen delivery in high-risk surgical patients [ISRCTN42445141]

INTRODUCTION: Preventing perioperative tissue oxygen debt contributes to a better postoperative recovery. Whether the beneficial effects of fluids and inotropes during optimization of the oxygen delivery index (DO(2)I) in high-risk patients submitted to major surgeries are due to fluids, to inotropes, or to the combination of the two is not known. We aimed to investigate the effect of DO(2)I optimization with fluids or with fluids and dobutamine on the 60-day hospital mortality and incidence of complications. METHODS: A randomized and controlled trial was performed in 50 high-risk patients (elderly with coexistent pathologies) undergoing major elective surgery. Therapy consisted of pulmonary artery catheter-guided hemodynamic optimization during the operation and 24 hours postoperatively using either fluids alone (n = 25) or fluids and dobutamine (n = 25), aiming to achieve supranormal values (DO(2)I > 600 ml/minute/m(2)). RESULTS: The cardiovascular depression was an important component in the perioperative period in this group of patients. Cardiovascular complications in the postoperative period occurred significantly more frequently in the volume group (13/25, 52%) than in the dobutamine group (4/25, 16%) (relative risk, 3.25; 95% confidence interval, 1.22–8.60; P < 0.05). The 60-day mortality rates were 28% in the volume group and 8% in the dobutamine group (relative risk, 3.00; 95% confidence interval, 0.67–13.46; not significant). CONCLUSION: In patients with high risk of perioperative death, pulmonary artery catheter-guided hemodynamic optimization using dobutamine determines better outcomes, whereas fluids alone increase the incidence of postoperative complications

Prognostic factors for patients treated with abiraterone

© Cecilia Melo Alvim. This work is licensed under the Creative Commons Attribution4.0 License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/Aim: To evaluate prostate-specific antigen response (PSAr) defined as a ≥50% decrease in PSA concentration from the pretreatment value, as a prognostic factor in patients with metastatic castration-resistant prostate cancer (mCRPC) treated with abiraterone acetate (AA). Methods: Retrospective evaluation of patients with mCRPC treated with AA. Results: 124 patients were identified. Median overall survival and progression-free survival for patients achieving PSAr versus patients without PSAr were 29.3 versus 9.7 months and 17.0 versus 5.2 months, respectively. Multivariate analysis confirmed that PSAr correlated with better overall survival (hazard ratio: 0.19; 95% CI: 0.10-0.38; p < 0.001) and progression-free survival (hazard ratio: 0.24; 95% CI: 0.14-0.41; p < 0.001). Conclusion: PSAr can be utilized as prognostic and predictive factors in mCRPC patients treated with AA.info:eu-repo/semantics/publishedVersio

Identification and Validation Model for Informative Liquid Biopsy-Based microRNA Biomarkers:Insights from Germ Cell Tumor In Vitro, In Vivo and Patient-Derived Data

Liquid biopsy-based biomarkers, such as microRNAs, represent valuable tools for patient management, but often do not make it to integration in the clinic. We aim to explore issues impeding this transition, in the setting of germ cell tumors, for which novel biomarkers are needed. We describe a model for identifying and validating clinically relevant microRNAs for germ cell tumor patients, using both in vitro, in vivo (mouse model) and patient-derived data. Initial wide screening of candidate microRNAs is performed, followed by targeted profiling of potentially relevant biomarkers. We demonstrate the relevance of appropriate (negative) controls, experimental conditions (proliferation), and issues related to sample origin (serum, plasma, cerebral spinal fluid) and pre-analytical variables (hemolysis, contaminants, temperature), all of which could interfere with liquid biopsy-based studies and their conclusions. Finally, we show the value of our identification model in a specific scenario, contradicting the presumed role of miR-375 as marker of teratoma histology in liquid biopsy setting. Our findings indicate other putative microRNAs (miR-885-5p, miR-448 and miR-197-3p) fulfilling this clinical need. The identification model is informative to identify the best candidate microRNAs to pursue in a clinical setting

Direct detection and measurement of wall shear stress using a filamentous bio-nanoparticle

The wall shear stress (WSS) that a moving fluid exerts on a surface affects many processes including those relating to vascular function. WSS plays an important role in normal physiology (e.g. angiogenesis) and affects the microvasculature's primary function of molecular transport. Points of fluctuating WSS show abnormalities in a number of diseases; however, there is no established technique for measuring WSS directly in physiological systems. All current methods rely on estimates obtained from measured velocity gradients in bulk flow data. In this work, we report a nanosensor that can directly measure WSS in microfluidic chambers with sub-micron spatial resolution by using a specific type of virus, the bacteriophage M13, which has been fluorescently labeled and anchored to a surface. It is demonstrated that the nanosensor can be calibrated and adapted for biological tissue, revealing WSS in micro-domains of cells that cannot be calculated accurately from bulk flow measurements. This method lends itself to a platform applicable to many applications in biology and microfluidics

XIST-promoter demethylation as tissue biomarker for testicular germ cell tumors and spermatogenesis quality

Background: The event of X chromosome inactivation induced by XIST, which is physiologically observed in females, is retained in testicular germ cell tumors (TGCTs), as a result of a supernumerary X chromosome constitution. X chromosome inactivation also occurs in male germline, specifically during spermatogenesis. We aimed to analyze the promoter methylation status of XIST in a series of TGCT tissues, representative cell lines, and testicular parenchyma. Methods: Two independent cohorts were included, comprising a total of 413 TGCT samples, four (T)GCT cell lines, and 86 testicular parenchyma samples. The relative amount of methylated and demethylated XIST promoter fragments was assessed by quantitative methylation-specific PCR (qMSP) and more sensitive high-resolution melting (HRM) methylation analyses. Results: Seminomas showed a lower amount of methylated XIST fragments as compared to non-seminomas or normal testis (p < 0.0001), allowing for a good discrimination among these groups (area under the curve 0.83 and 0.81, respectively). Seminomas showed a significantly higher content of demethylated XIST as compared to non-seminomas. The percentage of demethylated XIST fragment in cell lines reflected their chromosomal constitution (number of extra X chromosomes). A novel and strong positive correlation between the Johnsen’s score and XIST demethylation was identified (r = 0.75, p < 0.0001). Conclusions: The X chromosome inactivation event and demethylated XIST promoter are promising biomarkers for TGCTs and for assessing spermatogenesis quality

Influence of the calcium concentration in the presence of organic phosphorus on the physicochemical compatibility and stability of all-in-one admixtures for neonatal use

<p>Abstract</p> <p>Background</p> <p>Preterm infants need high amounts of calcium and phosphorus for bone mineralization, which is difficult to obtain with parenteral feeding due to the low solubility of these salts. The objective of this study was to evaluate the physicochemical compatibility of high concentrations of calcium associated with organic phosphate and its influence on the stability of AIO admixtures for neonatal use.</p> <p>Methods</p> <p>Three TPN admixture formulas were prepared in multilayered bags. The calcium content of the admixtures was adjusted to 0, 46.5 or 93 mg/100 ml in the presence of a fixed organic phosphate concentration as well as lipids, amino acids, inorganic salts, glucose, vitamins and oligoelements at pH 5.5. Each admixture was stored at 4°C, 25°C or 37°C and evaluated over a period of 7 days. The physicochemical stability parameters evaluated were visual aspect, pH, sterility, osmolality, peroxide formation, precipitation, and the size of lipid globules.</p> <p>Results</p> <p>Color alterations occurred from the first day on, and reversible lipid film formation from the third day of study for the admixtures stored at 25°C and 37°C. According to the parameters evaluated, the admixtures were stable at 4°C; and none of them presented precipitated particles due to calcium/phosphate incompatibility or lipid globules larger than 5 μm, which is the main parameter currently used to evaluate lipid emulsion stability. The admixtures maintained low peroxide levels and osmolarity was appropriate for parenteral administration.</p> <p>Conclusion</p> <p>The total calcium and calcium/phosphorus ratios studied appeared not to influence the physicochemical compatibility and stability of AIO admixtures.</p

Evidence for Reductive Genome Evolution and Lateral Acquisition of Virulence Functions in Two Corynebacterium pseudotuberculosis Strains

Ruiz JC, D'Afonseca V, Silva A, et al. Evidence for Reductive Genome Evolution and Lateral Acquisition of Virulence Functions in Two Corynebacterium pseudotuberculosis Strains. PLoS ONE. 2011;6(4): e18551.Background: Corynebacterium pseudotuberculosis, a Gram-positive, facultative intracellular pathogen, is the etiologic agent of the disease known as caseous lymphadenitis (CL). CL mainly affects small ruminants, such as goats and sheep; it also causes infections in humans, though rarely. This species is distributed worldwide, but it has the most serious economic impact in Oceania, Africa and South America. Although C. pseudotuberculosis causes major health and productivity problems for livestock, little is known about the molecular basis of its pathogenicity. Methodology and Findings: We characterized two C. pseudotuberculosis genomes (Cp1002, isolated from goats; and CpC231, isolated from sheep). Analysis of the predicted genomes showed high similarity in genomic architecture, gene content and genetic order. When C. pseudotuberculosis was compared with other Corynebacterium species, it became evident that this pathogenic species has lost numerous genes, resulting in one of the smallest genomes in the genus. Other differences that could be part of the adaptation to pathogenicity include a lower GC content, of about 52%, and a reduced gene repertoire. The C. pseudotuberculosis genome also includes seven putative pathogenicity islands, which contain several classical virulence factors, including genes for fimbrial subunits, adhesion factors, iron uptake and secreted toxins. Additionally, all of the virulence factors in the islands have characteristics that indicate horizontal transfer. Conclusions: These particular genome characteristics of C. pseudotuberculosis, as well as its acquired virulence factors in pathogenicity islands, provide evidence of its lifestyle and of the pathogenicity pathways used by this pathogen in the infection process. All genomes cited in this study are available in the NCBI Genbank database (http://www.ncbi.nlm.nih.gov/genbank/) under accession numbers CP001809 and CP001829

Rate and duration of hospitalisation for acute pulmonary embolism in the real-world clinical practice of different countries : Analysis from the RIETE registry

publishersversionPeer reviewe

Worldwide comparison of survival from childhood leukaemia for 1995–2009, by subtype, age, and sex (CONCORD-2): a population-based study of individual data for 89 828 children from 198 registries in 53 countries

Background Global inequalities in access to health care are reflected in differences in cancer survival. The CONCORD programme was designed to assess worldwide differences and trends in population-based cancer survival. In this population-based study, we aimed to estimate survival inequalities globally for several subtypes of childhood leukaemia. Methods Cancer registries participating in CONCORD were asked to submit tumour registrations for all children aged 0-14 years who were diagnosed with leukaemia between Jan 1, 1995, and Dec 31, 2009, and followed up until Dec 31, 2009. Haematological malignancies were defined by morphology codes in the International Classification of Diseases for Oncology, third revision. We excluded data from registries from which the data were judged to be less reliable, or included only lymphomas, and data from countries in which data for fewer than ten children were available for analysis. We also excluded records because of a missing date of birth, diagnosis, or last known vital status. We estimated 5-year net survival (ie, the probability of surviving at least 5 years after diagnosis, after controlling for deaths from other causes [background mortality]) for children by calendar period of diagnosis (1995-99, 2000-04, and 2005-09), sex, and age at diagnosis (< 1, 1-4, 5-9, and 10-14 years, inclusive) using appropriate life tables. We estimated age-standardised net survival for international comparison of survival trends for precursor-cell acute lymphoblastic leukaemia (ALL) and acute myeloid leukaemia (AML). Findings We analysed data from 89 828 children from 198 registries in 53 countries. During 1995-99, 5-year agestandardised net survival for all lymphoid leukaemias combined ranged from 10.6% (95% CI 3.1-18.2) in the Chinese registries to 86.8% (81.6-92.0) in Austria. International differences in 5-year survival for childhood leukaemia were still large as recently as 2005-09, when age-standardised survival for lymphoid leukaemias ranged from 52.4% (95% CI 42.8-61.9) in Cali, Colombia, to 91.6% (89.5-93.6) in the German registries, and for AML ranged from 33.3% (18.9-47.7) in Bulgaria to 78.2% (72.0-84.3) in German registries. Survival from precursor-cell ALL was very close to that of all lymphoid leukaemias combined, with similar variation. In most countries, survival from AML improved more than survival from ALL between 2000-04 and 2005-09. Survival for each type of leukaemia varied markedly with age: survival was highest for children aged 1-4 and 5-9 years, and lowest for infants (younger than 1 year). There was no systematic difference in survival between boys and girls. Interpretation Global inequalities in survival from childhood leukaemia have narrowed with time but remain very wide for both ALL and AML. These results provide useful information for health policy makers on the effectiveness of health-care systems and for cancer policy makers to reduce inequalities in childhood survival